Capture, concentration and quantitation of abnormal prion protein from biological fluids using depth filtration

ABSTRACT

Methods for producing biological solutions such as immunoglobulins and in particular anti-D immunoglobulin substantially free of abnormal prion protein resulting therefrom. Specifically provided are methods for aggregation of prions and depth filtration of the biological solution to capture and remove abnormal and if desired, normal prion protein. The prion protein may then be eluted from the depth filter and filter washes and concentrated sufficient for detection at limits currently required by available assays.

BACKGROUND OF THE INVENTION

[0001] Transmissible spongiform encephalopathies (TSEs) are a collectionof neurodegenerative diseases characterized by progressive dementia,ataxia, amyloid plaque formation and spongiform degeneration in thecentral nervous system (CNS) (Prusiner, S. B., 1993, Dev. Biol. Stand.80, 31-44). The causative agent in such diseases is now understood to beabnormal prion protein. The fundamental event in TSEs such asCreutzfeldt-Jakob disease (CJD) in humans, bovine spongiformencephalopathy (BSE) is cattle and scrapie in sheep is the conversion ofthe normal cellular prion protein PrP^(C), into a pathogenic isoform,PrP^(sc). Accumulation of PrP_(sc) in the brain of prion-infectedanimals correlates with the rise in titer of infectious prions and isused as a diagnostic marker for prion diseases. In light of the threatof an interspecies transmission of BSE to humans, a large number ofdomestic animals must be tested for the presence of PrP_(sc) in thebrain or other suitable material. In the absence of covalentmodifications that would allow a distinction between PrP^(sc) andPrP^(C), PrP^(sc) is routinely detected in Proteinase K (PK)-treatedhomogenates by Western blotting or enzyme-linked immunosorbent assay(ELISA) utilizing the fact that PrP^(sc) but not PrP^(c) is partiallyprotease resistant. Notably, these currently available assays do nottake advantage of the fact that PrP^(sc) forms aggregates. It is nowbelieved that formation of detergent-resistant PrP^(sc) aggregates is ageneral biochemical property of PrP^(sc) even for rare prion strainswhere PrP_(sc) is sensitive to proteolytic digestion. This aggregationoccurs when the prions are exposed to an aggregation aid for exampleincluding a complexing agent.

[0002] The fatal human neurodegenerative disorder CJD has also beentransmitted iatrogenically via a number of routes suggesting thepossibility that the causative agent might also be transmitted via bloodproducts. The identification of a new form of human TSE, named “variant”CJD (vCJD), confirmation of an association with the agent of bovinespongiform encephalopathy (BSE) and evidence that the distribution ofthe agent of vCJD in human tissues may differ from that of classical CJDsuggests the existence of a theoretical risk that blood or bloodproducts may transmit PrP^(sc) (see Turner et al., Blood Reviews 1998;12:255-68).

[0003] A number of blood products are prepared for medical use frompooled donations of human plasma including normal and specificimmunoglobulins, coagulation factor concentrates and solutions ofalbumin. There is currently considerable concern about the possibilitythat biopharmaceutical products from human or animal sources maytransmit TSEs. Human plasma proteins for parenteral administrationinherently carry a risk for disease transmission. Current technology forplasma screening and process steps for the removal or inactivation ofviruses has greatly improved the safety of these products, see BurnoufT, et al., Blood Reviews 2000; 14:94-110, in this regard. However,suitable screening tests have not yet been developed for abnormalPrP^(sc), which are also extremely resistant to chemical and physicalmeans of inactivation. To determine the probability of vCJD having beentransmitted to patients by products derived from this plasma, it isnecessary to determine the transmissibility of the PrP^(sc) inclinically relevant circumstances, the extent to which procedures usedfor plasma fractionation were capable of eliminating the PrP^(sc) fromplasma products, and the extent to which the agent PrP^(sc) can bedetected in the biological product using available assays.

[0004] Human plasma is obtained from whole blood following removal ofthe larger cellular fractions. Recent studies performed by the plasmafractionation industry have demonstrated that process steps used in themanufacture of human plasma products may reduce PrP^(sc) (see Foster P.,Trans. Med. 1999, 9:3-14; Lee DC et al., J. Virol. Methods 2000,84:77-89; Foster P. et al., Vox Sang 2000, 78:86-95, and Lee D. et al.,Transfusion 2001, 41:449-55.) These process steps include Cohnfractionation, depth filtration and chromatography. Foster et al. (VoxSang, supra.) demonstrated that depth filtration was effective inremoving significant amounts of abnormal prion protein (PrP^(sc)) fromboth immunoglobulin and albumin.

[0005] There is therefore a need to develop methods of capture andremoval of the abnormal infective prions from animal or human derivedmedicinal products or food products which are effective yet do notsubstantially degrade and/or remove the biological activity or foodvalue of the product. Due to the limitations of the current methods ofdetection and quantitation of abnormal prions, there is an unmet need inability to concentrate to above detection limits and thereafter detectand accurately quantitate the abnormal prion protein (PrP^(sc)) from thesample.

[0006] The instant invention is based on the surprising discovery thatdepth filtration of aqueous liquids containing biological products, suchas for example a biologically active protein, with one or more depthfilters having a pore size less than six microns, is surprisinglyeffective in removing abnormal infective prion proteins. Moreparticularly, these inventors have made the surprising discovery thatdepth filtration of aqueous liquids containing biological products, suchas for example a biologically active protein, with one or more depthfilters having a pore size less than six microns, after treatment withan aggregation aid, is surprisingly effective in removing abnormalinfective prion proteins.

[0007] The invention provides a method for the capture, removal,concentration and subsequent accurate quantitation of PrP^(sc)associated with TSEs, when such TSEs are contained in biological or foodproducts.

[0008] In particular, the invention provides a method for said capture,removal, concentration and subsequent accurate quantitation of PrP^(sc)associated with TSEs, in biologicals that have been treated with one ormore aggregation aids which results in aggregation of the PrP^(sc) suchthat the PrP_(sc) will be captured in and on a filter. Any method thatresults in such aggregation may be employed as an aggregation aid ascontemplated herein. In particular it has been found that solvents suchas for example alcohols may be employed. In the methods of theinvention, an aggregation aid such as a solvent liquid that has beenadmixed with the biological or food product is passed through a filterformed of a matrix of cellulose fiber impregnated with diatomaceousearth or similar filter material which may be coated with a cationicresin having an average pore diameter of the filter ranging from 0.1micron to 6 micron. Typically the filter may be a single use disposablefilter.

[0009] In particular, the invention provides a method for the capture,removal, concentration and subsequent accurate quantitation of PrP^(sc)associated with TSEs in biologicals that have been treated with one ormore aggregation aids, for example solvent such as for example analcohol, such as for example alcohol-fractionated immunoglobulinsolutions, which comprises passing the solvent liquid containing thebiological or food product through a depth filter formed of a matrixcomprising solid particles of porous material and having a pore sizeproviding a retention less than 6 μm. Typically the filter will be asingle use disposable filter. The treatment with the aggregation aid(s)may be accomplished with the one or more aids admixed together or usedin series.

[0010] By the terms “removal” or “capture” is meant the actual physicalremoval of the PrP^(sc) from the liquid containing the desired protein.For practical purposes, the recovery of the desired protein in itsoriginal biological state should be substantially maintained at least toa level in excess of 50′, preferably 80%, more preferably >90%.

[0011] Using the methods of the invention, removal of the abnormalinfective prion protein may be achieved to an extent of at least10^(2.5), 10³, preferably 10⁴, more particularly >10⁵.

[0012] Aside from removal of the infective PrP^(sc) from the biologicalor food product, the invention also relates to the elution from the oneor more filters and subsequent concentration of the captured and elutedPrP^(sc) using an elution buffer which may comprise, for example,hypertonic solutions such as for example high salt solutions so thePrP_(sc) may be accurately quantitated using available assays.

[0013] Thus, the instant invention provides for aggregation of prionsfollowed by filtration for the purification of a biological or foodsolution, the elution of the prions from the filter and theconcentration of the PrP^(sc) so as to enable one skilled in the art toemploy available assays to quantitate both total prion and PrP^(sc) in abiological or food sample. The invention will further allow the rapidhigh-throughput testing of large numbers of samples for PrP^(sc).

[0014] The invention also relates to the treated biological or foodsolution.

[0015] Since the source of human plasma is whole blood following removalof the larger cellular fractions, we therefore, in order to simulate thestate expected of a TSE agent in plasma for fractionation, herein usedas an inoculum a fraction of scrapie-infected hamster brain from whichintact cells and larger fragments had been removed. TSE diseases arebelieved to be transmitted either by protease -K-resistant,conformationally abnormal prion protein (PrP^(sc)). We herein disclosean in vitro method of analysis to determine the distribution ofhamster-adapted scrapie PrP^(sc) as a marker for the partitioningbehavior of vCJD.

[0016] TSE agents are highly resistant to inactivation, thereforereduction of any product-associated risk will be dependent on thephysical removal of infective material during product manufacture.Process technologies used in the manufacture of plasma products includethe separation of proteins by precipitation and chromatography withresultant protein solutions being clarified and sterilized by depth andmembrane filtration procedures, respectively. Some of these technologiesby their modification with the methods of this invention, may be capableof removing TSE agents from a product stream.

[0017] PrP protein was detected herein using a Western Blot with themonoclonal antibody 3F4 specific for hamster PrP. This antibody reactswith residues 109-112 PrP from only humans, hamsters and felines.Incubation with 3F4 antibody was at a concentration of 0.6 ug/ml for aminimum of 1 hour, after which excess antibody was washed away and themembranes incubated with a rabbit anti-mouse horseradish peroxidaseconjugate (1:1000 dilution) for a minimum of 1 hour. After extensivewashing with TTBS, the membranes were developed using enhancedchemiluminescence.

[0018] In the manufacture of RhoGAM® RHO(D) Immune Globulin (Human) bythis Assignee, PrP^(sc) was removed to the limit of detection duringdepth filtration steps that are also used in the manufacture ofimmunoglobulins.

[0019] Western blotting is a method used to identify and characterizePrP^(sc). The PrP^(sc) is isolated by extraction and is differentiatedby its partial resistance to proteinase K digestion. The PrP^(RES)(PrP^(sc) resistant to proteinase digestion) is identified by themigration positions of the glycosylation forms and fragments. Thesensitivity of this assay is approximately 3 logs less sensitive thanthe infectivity assay. This sensitivity issue is partially overcome bycentrifuging the enzyme digested preparation, removing the supernatantand resuspending the prion material in a smaller volume, resulting in aconcentration of the prion material. We have shown that the prions canbe easily concentrated by filtering them through a filter aftertreatment with an aggregation aid, and later collected in a small volumeby elution. This technique can be used on a large scale to remove prionsfrom a product stream.

[0020] This procedure will have a major impact on the use of the Westernblot and indeed any other prion detection assay, to determine thepresence of PrP^(sc) in a biological matrix. This invention allows theTSE material to be quantitatively concentrated quickly to allow forenhanced detection. When seeking to purify a biological, food orcosmetic solution of PrP^(sc), this invention has the advantage in theease in which the biological, food or cosmetic solution filters throughthe large nominal pore size of the filter.

[0021] The methods of the invention are useful for the treatment ofbiologicals, foods and cosmetics by removing, eluting and, further,quantitating PrP^(sc), and depending on the aggregation aid(s) employed,PrP^(C). Among the biologicals that can be so treated are blood andblood components such as whole blood, blood serum and plasma, urine,cerebrospinal fluid and blood-derived biological products such asantibodies and immunoglobulins. One such antibody is the IgGimmunoglobulin known as monoclonal anti-D immunoglobulin or RhoGAM®Rho(D) Immune Globulin (Human). This polyclonal immunoglobulin is usedin the prevention of hemolytic disease of newborn wherein the mother isinjected with Rho(D) immunoglobulin of human origin. Such a product isRhoGAM®, available from the assignee hereof, and it operates bypreventing the unimmunized Rho (D) negative mother from responding toRho (D) antigen present on red cells and ‘received’ from an Rho(D)positive infant. Thus, by preventing anti-Rho (D) production by themother, the subsequent Rho (D) positive infant of this mother isprotected from hemolytic disease of the newborn. This successful productis currently produced by a Cohn alcohol fractionation type process.

[0022] RhoGAM® Rho(D) Immune Globulin (Human) was the first successfulprophylactic use of specific antibody to achieve antibody mediatedimmune suppression. RhoGAM® is an IgG immunoglobulin solution containinganti-Rho(D) at a dose of 300 micrograms of anti-D activity per dose.RhoGAM® can be given to the nonimmunized, Rho(D) negative pregnant womanat the appropriate time prevent future disease in her Rho(D) positiveoffspring. The disease is called hemolytic disease of the newborn ormore specifically, Rh-erythroblastosis fetalis.

[0023] A smaller dose of anti-Rho(D), MICRhoGAM® Rho(D) Immune Globulin(Human) Micro-Dose (50 micrograms of anti-Rho(D)) is also sold by theAssignee hereof for treatment of women who have abortions andmiscarriages at twelve weeks gestation or earlier. While the full doseprotects the recipient for up to 15 ml of Rho(D) positive red cells, thesmaller dose provides protection up to 2.5 ml of Rho(D) positive redcells. RhoGAM® is used as antenatal prophylaxis at 26 to 28 weeksgestation. Other indications include threatened abortion at any stage ofgestation with continuation of pregnancy, abortion or termination ofpregnancy at or beyond 13 weeks gestation, abdominal trauma or geneticamniocentesis, chorionic villus sampling (CVS) and percutaneousumbilical blood sampling (PUBS).

[0024] Most immunoglobulin injectable materials approved for use by theFDA and Bureau of Biologics have been produced by the alcoholfractionation procedure developed by Dr. E. Cohn of Harvard during the1940s and described in Cohn et al., J. Am. Chem. Soc. 68, 459 (1946),incorporated herein by reference. This procedure coupled with thecareful selection of plasma negative for hepatitis infectivity, HIV, andother blood-borne pathogens determined by the most sensitive testsavailable. That the products produced by this procedure are indeed safecan easily be demonstrated by the millions of non-infected recipients ofproduct. The inventors hereof have now found that the alcohol employedin the Cohn process referenced hereinabove is sufficient to act as anaggregation aid in that it causes sufficient numbers of PrP^(sc)particles to aggregate, such that PrP^(sc) can be removed to the limitsof detection using the inventive depth filtration, and eluted andconcentrated to a level sufficient for such detection.

[0025] The solvent composition employed has minimal effect on the IgGparticle but sufficiently aggregates the PrP^(sc) sufficient to enableit to be removed to below its level of detection using available assays.

[0026] It is therefore an object of this invention to provide a methodfor removal of PrP^(sc) and if desired, PrP^(C), from biological andfood solutions using prion aggregation aids and membrane or depthfiltration. Depth filtration is preferably used.

[0027] It is also an object of the invention to remove PrP^(sc) and ifdesired, PrP^(C), from protein-containing liquids, particularly thosederived from human plasma, without unacceptable effects on the nature orbiological activity of the proteins.

[0028] It is a further object of the invention to capture, concentrateand detect to accurate quantitation, PrP^(sc) from any biological fluidusing the methods disclosed herein.

[0029] It is an object of the instant invention to provide abnormalinfective prion -cleared, pure immunoglobulin for injection. Such asubstantially pure product is produced using the processing methods ofthe invention.

[0030] It is a further object of this invention to provide amanufacturable process for purifying immunoglobulins from abnormalinfective prion which is reasonable in terms of temporal, square footand protein yield requirements.

[0031] It is a further object of the invention to provide a depth filterwhich can be a single use filter and may be disposed of having removedPrP^(sc) from the process stream.

[0032] It is a further object of this invention to provide aconcentrated PrP^(sc) solution, by elution of said prions from the depthfilter and filter washes.

[0033] It is yet a further object of this invention to provide a rapidassay for the assessment of PrP^(sc) in various biological materialsincluding biological fluids and human blood and plasma-derived products.Use of such assays as, for example, the Western Blot, require sufficientlevels of prions unavailable in non-prion-aggregated, non-filteredbiological solutions. This method provides a practical method tocapture, elute and concentrate prions so that they can be detected usingcurrently available assays. Use of these novel capture and elutionmethods increases sensitivity about 3 logs, enabling reduction in thevolumes needed to perform the detection assays.

SUMMARY OF THE INVENTION

[0034] The methods of this invention are used to produce immunoglobulin(preferably monoclonal) substantially purified of abnormal prionprotein. The substantially purified immunoglobulin is for examplemonoclonal or polyclonal anti-D immunoglobulin, for example RhoGAM® orMICRhoGAM®. This immunoglobulin formulation comprises from about 4.0 to6.0% immunoglobulin by weight, and from about 80 to 200 ppm polysorbate80, more preferably about 5.0% immunoglobulin by weight, and about 130ppm polysorbate 80.

[0035] The above referenced immunoglobulin formulation is made generallyby the steps of fractionating human plasma using an aggregation aid suchas for instance an alcohol, wherein the fractionation comprises afiltration step; resuspending the resulting Precipitate II; admixing theresuspended Precipitate II with a high ionic strength buffer containingan excipient; and performing nanofiltration on the immunoglobulin.

[0036] The alcohol is preferably methanol and the filtration step isperformed on Supernatant III in the fractionation process, using a depthfilter for instance a Cuno Zeta Plus 90S depth filter.

[0037] The methods disclose a process for the manufacture of anti-Dantibody substantially purified of abnormal prion protein, includingfractionating human plasma in the presence of an aggregation aid such asfor instance an alcohol wherein the fractionation comprises a filtrationstep. The filtration step may employ a depth filter such as for instancea Cuno Zeta Plus 90S depth filter, having a pore size rating of fromabout 0.6 to 6 micron. The resultant supernatant, referred to in theprocess as “Supernatant III” is processed to form a precipitate (calledin the method “Precipitate II”), which is then resuspended and admixedwith processing aids and nanofiltration on the resulting anti-D antibodyperformed thereon. The processing aids may include a high ionic strengthbuffer and a non-ionic excipient, for example 150 mM NaCl-Glycine bufferand polysorbate 80.

[0038] Further disclosed herein is a process for the manufacture ofbiological product substantially purified of abnormal prion protein byadmixing the biological product with an aggregation aid such as asolvent sufficient to form aggregated abnormal and normal prion protein;and filtering the thusly acquired admixture with a depth filter. Thebiological product is blood or blood product, cerebrospinal fluid, orurine. When the product is blood, the blood may first be clinicallycentrifuged and the red blood cells and platelets removed from the bloodprior to admixing with the aggregation aid. After the filtering step,the red blood cells and platelets may be added back to the blood. Thedepth filter may include for example a Cuno Zeta Plus 90S depth filter.The aggregation aid may be a solvent such as for instance an alcohol,for instance ethanol or methanol at a concentration of from about 2% toabout 100%.

[0039] Yet further disclosed herein is a method for quantitatingabnormal prion protein in a biological solution. This method maycomprise admixing the biological solution with an aggregation aid(s)such as a solvent sufficient to aggregate the abnormal prion protein,filtering the admixture with a depth filter, eluting the abnormal prionprotein off the depth filter by washing the filter with an elutionbuffer, optionally concentrating the elution buffer by such method ascentrifugation, and performing an assay for abnormal prion protein onthe elution buffer. The biological solution may be blood or a bloodproduct (for example an immunoglobulin), cerebrospinal fluid, or urine.

BRIEF DESCRIPTION OF THE DRAWING

[0040]FIG. 1 is a flow sheet showing the process of fractionation ofhuman plasma to obtain anti-Rh globulin. During this fractionationprocess the material may be filtered to capture prion protein.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The instant invention employs one or more aggregation aids toaggregate prion in a fluid, such that prion (normal and/or abnormal) maybe eluted, captured, concentrated and detected. One class of aggregationaids will aggregate both abnormal infective prion (PrP^(sc)) as well asnormal prions in a fluid, such as a biological fluid, which prionaggregates may then be removed, eluted, concentrated and eitherabnormal, normal or both types of prions accurately quantitated usingthe methods of the invention.

[0042] The invention further contemplates use of an aggregation aidwhich is a complexing agent, which agent aggregates either normal prionsor abnormal prions, depending on the properties of the complexing agent.Such complexing agents include metal ions such as for example Cu2+, Ni,Zn, and Ag.

[0043] The invention allows a filter such as for example a depth filterto be used even with biological fluids comprising globular proteinmolecules such as for example an immunoglobulin or antibody, withoutappreciable yield loss and no significant change in immunoglobulinsubclass, immunoglobulin aggregate level or immunoglobulin stability.

[0044] The methods of the invention yield a biological fluidsubstantially free of abnormal infective prions (PrP^(sc)) and ifdesired, normal prion (PrP^(C)). The methods of aggregation andfiltration of the invention in fact can, when aggregation aids areproperly selected to do so, ensure that all possible categories ofprion, both normal and abnormal, are removed from the product.

[0045] The process is in particular applicable to the treatment of wholeblood, blood components (e.g., serum, plasma), urine, CSF, or anybiological such as for example liquids containing albumin,immunoglobulins (for example, IgG) and fragments thereof, bloodcoagulation factors such as Factor IX, thrombin, fibronectin,fibrinogen, Factor VIII and Factor II, VII, IX, and X and other proteinsderived from plasma. It is also applicable to the treatment of plasma,Factor XI, Factor XIII, hemoglobin, alpha-2-macroglobulin, haptogobin,transferrin, apolipoproteins, protein C, protein S, C-1-esteraseinhibitor, enzymes (for example, streptokinase), inter-alpha-trypsininhibitor, growth hormones and Von Willebrand factor. Naturallyoccurring and recombinant analogues of the above may be treated. Inaddition, the invention is applicable to the treatment of other naturalproducts including foods, drinks, cosmetics etc. It is also applicableto other non-plasma animal-derived products, such as heparin andhormones.

[0046] As stated herein, the biological fluids that can be processedusing the methods of the present invention include blood and bloodcomponents such as whole blood and components thereof including bloodserum and blood plasma, urine, cerebrospinal fluid, and any biologicalproducts such as for example antibodies and immunoglobulins. The humanplasma treated and filtered in the instant invention can be obtained bythe fractionation methods of Cohn et al. (the “Cohn process”),referenced hereinabove, by batch or column exchange chromatography, orby affinity chromatography. In the method of producing immunoglobulin,particularly anti-D immunoglobulin such as RhoGAM Rho(D) Immune Globulin(Human), reference is made herein to commonly assigned U.S. Pat. No.6,096,872, issued Aug. 1, 2000, to Van Holten et al., the contents ofwhich are herein incorporated by reference.

[0047] Cohn, U.S. Pat. No. 2,390,074, the contents of which are hereinincorporated by reference, discloses a method of fractionating blood bywhich gamma globulins are prepared. The gamma globulins prepared by theCohn method contain 19 S globulin, plasminogen and lipids. While thisgamma globulin is eminently suitable for prophylaxis against diseasessuch as measles and tetanus, the presence of the 19 S globulin,plasminogen and lipids are unnecessary contaminants and may decrease itseffectiveness in preventing immunization to the Rh-factor on the fetalerythrocytes.

[0048] The substantially pure anti-Rh globulin manufactured by thevalidatable processes of the present invention is prepared from humanplasma which contains albumin, plasminogen, alpha, beta and gammaglobulins and various lipids. Specifically, the anti-Rh globulin of theinvention is a gamma globulin.

[0049] The fractionation of human plasma to obtain anti-Rh globulin iscarried out according to the methods of the aforementioned Cohn et al.,as well as commonly-assigned U.S. Pat. No. 3,449,314 to Pollack et al.,the teachings of which patents are hereby incorporated by referenceherein. With reference to the accompanying flow sheet of FIG. 1, theability to fractionate human plasma is dependent upon the solubility ofthe various components of the plasma. At each stage of thefractionation, the separation of the fraction and the ultimate removalof those components which are undesirable in the anti-Rh globulin aredetermined by the critical control of pH, temperature, concentration ofthe precipitant and the ionic strength of the system.

[0050] Various aggregation aids may be used in the aggregation of theprions resident in the biological fluids of the invention. There are anumber of classes of aggregation aids that can be used, all working onthe principle of changing the characteristics (e.g., the size) of theprion without aggregating or otherwise adversely affecting the milieucontaining it.

[0051] It will be appreciated that some aggregation aids aggregate bothabnormal and normal prion. Aggregation aids in this class includeorganic solvents of low dielectric constant such as acetone andalcohols, which are known to precipitate proteins and have been used inthe fractionation of plasma. More particularly, the organic solventsutilized in the method of this invention include the various alcoholswhich are completely water-miscible and those that do not react withproteins, such as for example ethanol, methanol, isopropyl, isopropanol,n-propanol, isopropyl ether, ketones, aldehydes, etc., and acetone, andpreferably methanol. Other similar aggregation aids in this class thatmay be used, to the extent they are compatible with the biologicalmaterial being treated, include ammonium sulfate, caprylic acid, and thechemical agents trichloroacetic acid (TCA), dialdehydes, heteropolyacids, and lactate monohydrate C₁₈H₂₁N₃O₄H₂O.

[0052] It will further be appreciated that some aggregation aids willaggregate the abnormal prion thereby allowing it to be removed, whileleaving the normal prion in its native state, and vice versa—this classof aggregation aids are the complexing agents. These complexing agentsbind to the prion protein and include heteropolymolybdates,heteropolytungstates, sodium phosphotungstate (NaPTA) (all of whichaggregate only abnormal prion), and the biological agents such asantibodies (monoclonal or polyclonal), the antibodies having actiondependent upon their specificities, enzymes (such as for exampleplasminogen (which aggregates only abnormal prion) and peptides,peptides having selective action dependent upon their composition. Afurther aggregation aid which is a complexing agent includes the metalion Cu2+, which aggregates normal prion. Other similar metal ions mayinclude Ni, Zn, and Ag. These agents can be employed as a prion capturemechanism when bound to a substrate. In one embodiment it iscontemplated that the complexing agents may be used in series, forinstance, the ion Cu2+may be admixed with the biological solution andthe normal prions removed by filtration, followed by admixing theresulting biological solution filtrate with the NaPTA to complex theabnormal prions, which may then be captured, eluted and concentrated anddetected using known assay methods.

[0053] The aggregation aid methanol is preferred for prion removal fromimmunoglobulin solutions due to its comparatively lower toxicity andsafer handling (e.g., explosion danger) than other organic solvents.When such solvents are used they are generally present in the admixturewith the biological fluid in concentrations of about 2% to about 100% byvolume of biological. The concentration of the solvent is dictated inthe lower range by the minimum concentration required to aggregate theprions, and at the higher range by the integrity of the biological andthe filter media.

[0054] It has now been found by these inventors that the inventiveprocessing with aggregation aids such as those named hereinabove resultsin the aggregation of the prion (either or both PrP^(sc) and PrP^(C),depending on the aggregation aids and methods used in employing them)protein, and that using the methods of this invention, such aggregatescan be removed using filtration. Then, using the inventive methods offiltration and later elution from the filter and the filter washes, andif desired, concentrating the eluate, the PrP^(sc) can be obtained inconcentrations sufficient to enable accurate quantitation. Using themethods of the invention it has been found that such treatment issufficient to remove the PrP^(sc) from the immunoglobulin formulation tobelow its detection limits. The sensitivity of assays used to detectsuch prion is increased by approximately 3 logs, enabling the reductionin volume and therefore increasing prion concentration in the sample.

[0055] For the PrP^(sc) aggregation aspect of the invention, theaggregation aids that may be used are any that are found to precipitateabnormal infective prion protein fibrils while being compatible with thebiological materials being treated, and compatible with the filter beingused.

[0056] The filtration aspect of the invention may be carried out at anytemperature that is appropriate to the biological materials beingfiltered, indeed the conditions for filtration are mandated by thebiological, food or cosmetic product under filtration and not theconditions required to capture the prion material. In order to preventdenaturation of proteins during fractionation and filtration, thefractionation, where employed, and filtration may be preferably carriedout at low temperatures. Since protein solubility is temperaturedependent, the temperature chosen for each step of the fractionationmust be the lowest possible which permits the desired separation and/orfiltration in order to prevent denaturation. The pH conditions should bemandated by the liability of the biological product being purified.

[0057] The depth filters that may be used in the practice of the instantinvention are those depth filters that are either charged or uncharged.Examples of depth filters that may be utilized include Celite (WorldMinerals, Lompoc, Calif.), Millipore filters 75DE and SA (MilliporeCorporation, Bedford, Mass.), and Cuno 35P and Cuno Zeta Plus 90SP (CunoCorporation, Meriden, Conn.).

[0058] Most preferable for use in the instant invention are the 47 mmCuno Zeta Plus 90SP depth filter, along with the appropriate stainlesssteel filter housing, for small scale filtration work, and the 16 squarefoot cartridges used in manufacturing processes. Preferably the filterfor use in the practice of the instant invention is a depth filter,however non-depth filters may be employed for removal of aggregatedPrP^(sc) as long as the filtration through these filters does not resultin the clogging of said filters. Such alternate filters are for examplemembrane filters, charged or uncharged. Such filters include forexample, the disposable syringe filters Swinex or Millex 25 mm PVDFsyringe-driven filter units, 0.22 micron Opticap and Optiseal cartridges(Millipore Corporation, Bedford Mass.).

[0059] The pore size of the filters used in the practice of the removaland capture of PrP^(sc) (and where desired, PrP^(C)) aggregates of theinvention is relatively unimportant, however, the pore size can affectthe recovery of the biological product being filtered. The pore size ofthe filter matrix is preferably in the range of 0.2 to 6 microns,particularly 0.6 to 1.5 microns. The pore size is defined in terms ofthe particle size of particles retained thereon. Typically particles ofdefined size such as dextrans or microorganisms are used for calibrationpurposes.

[0060] The pore size of the filtering units employed in the productionof substantially pure, abnormal infective prion -free immunoglobulinproducts of the instant invention is less than about 6 μm, mostpreferably less than about 0.6 μm. However, any filter having a cutoffrating sufficient to reduce or eliminate abnormal infective prion from aproteinaceous solution can be employed in the processing methods of theinvention. For example, for depth filters, Cuno Zeta Plus 90S filterpads (Cuno Corporation, Meriden, Conn.) may be employed, such unithaving a molecular weight pore size rating of 0.1 to 5 micron.

[0061] Similarly, filter composition should have little effect on theability of the filters to capture the aggregated PrP^(sc), however,recovery of the prion material from said filters may require elutionbuffers such as high salt solutions or surfactants.

[0062] The filter material may comprise a depth filter which generallycomprises a self binding matrix of cellulose, together with a solidporous particulate material such as Kieselguhr, perlite or diatomaceousearth.

[0063] The depth filter generally has a thickness in the range 1-10 mm,particularly 2-5 mm. The material used for the depth filter should havelittle or no effect in the desirable protein concerned. Acceptable depthfilters include the Seitz KS80 filter of pore size 0.6 to 1.5 μm, theSeitz K200P, the Cuno Delipid Del 1 mini cartridge, effective filtrationarea 27 cm³, Millipore filters, particularly the Millipore CP20, alsoincluding conventional ultrafilters such as Millipore PTHKpolyethersulfone membrane, AMICON ym-100 regenerated celluloseultrafiltration membrane, (Millipore Corporation, Bedford Mass.), PALLFiltron Omega VR, Pall Ultrapore VFDV50 (Pall Corporation, East Hills,N.Y.), along with other cartridge filters, such as the Asahi Planovaregenerated cellulose cartridge filters, (Asahi, Tokyo Japan). Otherfilters that can be used are charged depth filters such as those of E.Begerow GmbH & Co., Langenlonsheim, Germany. However, the mostpreferable embodiments herein employ the Cuno Zeta Plus 90S filter pads,47 mm filter.

[0064] The flow rate of the biological material through the filters arethose rates suitable for ensuring proper filtration of the biologicalmaterial while not compromising the integrity of the filter or, in thecase of the biological material comprising large globular proteins, arate that does not compromise the structure of the proteins so as tomake the preparation unacceptable for its intended purpose. In the depthfiltration of an immunoglobulin product for example, filtration ratesrange from about 0.01 to about 20 ml/minute, more preferably about 10ml/minute, more preferably about 1 ml/minute.

[0065] The method may be carried out in the pH range of 4-10, preferably5-9, more preferably 6-8. However, the pH range will be determined bythat pH required to preserve the integrity of the biological beingtreated and the filter employed, and not by any limitation on theaggregation or filtration process itself.

[0066] The application of heat is unnecessary and the process can becarried out at substantially room temperature or below, in particular atthe temperatures of −5 to +20° C., as suitable for maintaining theintegrity of the biological and the filtering medium.

[0067] As stated hereinabove, an aspect of the instant invention is thetreatment of the biological fluid with an aggregation aid such as forexample a solvent sufficient to aggregate the prion contained therein soit may be captured by filtration and eluted in a concentrationsufficient for detection using known methods such as for example,Western Blot. Some biological fluids will be so treated as a function oftheir production, for example, immunoglobulins which are treated withalcohol in the Cohn process. Where the biological fluid is not alreadyso treated, it will be treated with a suitable aggregation aid such asthose stated hereinabove so as to aggregate the prions containedtherein. Such biological fluids are enumerated hereinabove and mayinclude blood and components thereof, urine and cerebrospinal fluid, aswell as immunoglobulins.

[0068] Following treatment of the biological with the aggregation aid,the filter pad is removed from its housing and prion eluted therefrom,concentrated if desired using a process such as centrifugation, andquantitated using available assays, all in accordance with remainingaspects of the invention, all herein described.

[0069] A protease resistant prion protein isoform is present in urine ofanimals and humans affected with prion disease. Shaked et al. (2001, J.Biol. Chem. 276 (34):31479-31482) discuss steps to isolate prions fromurine. The process described in Shaked et al. requires 2-15 ml of aurine sample to be sedimented for 5 minutes at 3000 rpm and thendialyzed overnight in cellulose membrane tubes. Subsequently the urinesamples were centrifuged at high speed (100,000×g) for 1 hour at 4° C.

[0070] The Shaked et al. procedure is time consuming and is limited bythe amount of sample that can easily be concentrated. Disclosed hereinis a procedure for accurately quantitating prions from a biologicalfluid such as urine, that may be accomplished in minutes and is notlimited by volume. During prion filtration and elution from urine withthe methods of the invention, up to 1 liter of urine volume may befiltered with a 47 mm Cuno filter. This volume difference allows for amagnitude increase in the concentrating capacity of the instantprocedure compared to the current state of the art. See Example 9herein.

[0071] When whole blood is used as the biological fluid in theinvention, a quantity of it, for example 1 liter, may first becentrifuged under conditions suitable for separating the cellularcomponent. The resulting plasma is admixed with an aggregation aid suchas for example a quantity of methanol, for instance in a ratio of about5 parts plasma to about 1 part methanol, to aggregate the prionmaterial. The admixture is gently mixed on a rotary shaker for a periodof time sufficient to aggregate the prions present, for example forabout one (1) minute. The admixture is then passed through a filter forexample a 47 mm Cuno Zeta Plus 90S filter. The material may then beeluted from the filter using an elution buffer as described herein, andthe prions quantitated by any suitable assay, such as for example aWestern Blot assay. If desired, the detection limit may be furtherimproved by including a PrP^(sc) sedimentation step. The samples werediluted and treated with Proteinase-K (PK) followed by AEBSF(4-(2-aminoethyl) benzensulfonyl fluoride) to inhibit proteinaseactivity. Following the PK treatment the sample is centrifuged at20,000×g for 1 hour at 4° C. The pellet is then prepared for SDS Page.

[0072] When the biological material to be treated with prion aggregationaids is an immunoglobulin, the biological material will be so treatedduring the plasma fractionation process. With reference to FIG. 1 andExample 1 herein, plasma units are pooled and then under specifiedconditions are centrifuged and relevant portions are retained forfurther processing with the aggregation aid, in this case, preferablymethanol. At the point of the fractionation wherein Supernatant III isobtained, the Supernatant III fraction is filtered using a membrane ordepth filter, which filtration removes the aggregated prions that mayhave been contained therein. The aggregated prions captured thereby maybe eluted from the filter and detected and quantitated using knownassays.

[0073] The capture and elution procedures of the invention result in anincrease in the detection limit of the assay by greater than 100 fold,now approaching the infectivity assay detection limit. Using methodscurrently available in the art, the infectivity assay can take months toyield results, dependent upon the species under study compared to hoursfor producing results using the methods of the invention.

[0074] Following aggregation of prions resident in biological fluid,whether by solvent or otherwise, the next steps are the prion (forexample, PrP^(sc)) elution and recovery. In these steps the filter orfilter pad(s) is/are removed and washed with elution buffer. One methodis the placement of the pad(s) in a receptacle such as for example apetri dish, a beaker or similar suitable container, a suitable volumefor example about 15 ml to about 100 ml of elution buffer added thereto,and the container placed on a rotary shaker at room temperature forabout 25 minutes. The filter-bound PrP^(sc) is thereby eluted therefromvia gentle washing with the elution buffer. Suitable elution buffersinclude any aqueous buffers, such as for example, hypertonic saltsolutions such as for example 1.0-2.0M NaCl buffers, sodiumacetate-methanol buffers at concentrations of 1.0M to about 2.0M.

[0075] If desired, the aggregated prions may be further concentrated bycentrifugation or any procedure known in 10 the art for achieving anincreased concentration.

[0076] Given the theoretical possibility for prion contamination ofblood products, it was especially important to elucidate theeffectiveness of depth filtration and the mechanism for prion removalfrom an intermediate from immunoglobulin production RhoGAM®Ultra-Filtered Rho(D) Immune Globulin (Human). In accomplishing thisgoal, these inventors used scrapie brain homogenate (SBH) fromscrapie-infected hamsters, as the source of the PrP^(sc). In order tocarry out such studies, the PrP^(sc) “spike” was first treated withdetergent to solubilize it, and sonicated to disrupt the fibrils. Thespike was treated so as to make the PrP^(sc) as small as possible so asto challenge the filtering system. The sonicated SBH was thensequentially 0.45, 0.22 and 0.1 micron membrane filtered to betterdefine the size of the PrP^(sc) spike prior to spiking. A previous study(Van Holten R, et al., Transfusion (submitted for publication)) haddemonstrated that this treatment did not adversely effect the PrP^(sc)and would additionally insure that the particles the depth filtrationwould remove would be closer in size to the individual fibrilsassociated with infection. A reduction in PrP^(sc) after depthfiltration could indicate that prion removal was due to the fibrilsadsorbing to the positively charged filter media, rather than bymechanical straining. The addition of the spike into the IgG diluted ina phosphate buffer/methanol mixture resulted in flocculation of thematerial which resulted in a cloudy appearance.

[0077] A Cuno Zeta Plus SP charged depth filter was used to filter theRhoGAM® Rho(D) Immune Gamma Globulin (Human) that was spiked with theSBH. Upon filtration through a Zeta Plus SP filter the cloudiness wasremoved. A layer of white precipitate was observed on the filter postfiltration. Upon Western blot analysis used to detect PrP^(RES) thefilter material was void of scrapie. With a 2.0M salt wash the prionmaterial was recovered from the filter. The Western Blot results areshown herein in Table 1.

[0078] Two control runs were also performed. In the first run, thePrP^(sc) spiked immunoglobulin intermediate was first filtered through a0.22 μm filter to insure that the PrP_(sc) did not aggregate to largerparticles that could be removed by the depth filter through mechanicalstraining. In the second run, the sonicated and filtered SBH was spikedinto Tris buffered saline (TBS) instead of the immunoglobulinintermediate, followed by the depth filtration.

[0079] The depth filter removed greater than four logs of PrP^(sc) fromthe filtrate of the immunoglobulin. A significant portion of thePrP^(sc) could be recovered from the immunoglobulin filtration byelution with high molarity NaCl solutions. The 0.22 μm prefiltration ofthe spiked Supernatant III removed all detectable PrP^(sc) prior todepth filtration. Less than one log of PrP^(sc) was removed from thebuffer control by depth filtration. See Examples 6 and 7.

[0080] It was thus found that depth filtration removed PrP^(sc) from theimmunoglobulin by mechanical straining rather than by adsorption to thefilter matrix. The immunoglobulin preparation caused the PrP^(sc) toaggregate from particles <0.1 μm in size to particles >0.22 μm, probablyas a result of the methanol in the immunoglobulin preparation. The depthfilter failed to remove PrP^(sc) from the buffer control sample.

[0081] In Example 3 herein, membrane filtration of the sonicated SBH wasperformed prior to depth filtration of the SBH spiked Supernatant III(“SupIII”) in order to insure that the depth filter would see particlesno greater than 0.1 micron in size. This would present the greatestchallenge to the depth filter and would allow characterization of themechanism of PrP_(sc) removal. The SBH was first sonicated to break upthe PrP^(sc) aggregates and facilitate the membrane filtration. Despitethe sonication, it was necessary to serially filter the SBH throughprogressively smaller filters (0.45 and 0.22 micron) to minimizeclogging of the 0.1 micron filter.

[0082] The Cuno Zeta Plus 90SP depth filter utilizes two mechanisms forparticle removal. Particles above the nominal pore size of approximately0.1 micron are retained predominately by mechanical straining. Below 0.1micron, particles with a negative charge are retained by electrokineticadsorption to the positively charged filter media (U.S. Pat. No.4,859,340). Since particles greater than 0.1 micron had been removedfrom the SBH prior to addition to the Supernatant III and subsequentdepth filtration, it appeared that the retention of the PrP^(sc) by thedepth filter was due to increase in particle size due to exposure tomethanol. However, the charge capture mechanism of removal would be ineffect when one departs from the isoelectric point of the prion beingcaptured.

[0083] Examination of the depth filter after filtration of the SBHspiked SupIII and prior to elution with the 1.0M and 2.0M NaCl solutionsrevealed a small amount of material on the surface of the depth filter.This was believed to be a precipitate formed when the SBH was added tothe SupIII, caused by the methanol present in the SupIII. In order todetermine if this precipitate contained PrP^(sc), a second run wasperformed where the SBH spiked SupIII was first pre-filtered through a0.22 micron filter prior to depth filtration. The pre-filtration removedPrP^(sc) to undetectable levels, indicating that in the prior run thePrP^(sc) was removed by precipitation and mechanical straining, ratherthan by electrostatic adherence to the depth filter. Prusiner et al.(Biochemistry 1980; 19:4883-91) demonstrated that ethanol readilyprecipitated PrP^(sc), so it is not surprising that the presence of themethanol used in this fractionation process would have the same effect.

[0084] In order to determine whether depth filtration would removePrP^(sc) in the absence of a precipitating alcohol, a control run wasperformed (see Example 4) where the PrP^(sc) was spiked into an aqueousbuffer and then depth filtered. The lack of removal of PrP^(sc) from thebuffer control indicated that the depth filter did not retain theprotein, either by mechanical means (because the PrP^(sc) had previouslypassed through a 0.1 micron filter) nor by electrostatic adherence.

[0085] These studies indicate that previous reports on the effectivenessof depth filtration to remove PrP^(sc) may be misleading. Indeed, depthfiltration does remove PrP^(sc), not by the absorptive mechanism usuallyassociated with depth filtration but by mechanical straining of theprecipitated protein. The results of this study indicate that depthfiltration alone is ineffective in removing PrP^(sc). However, when usedin conjunction with a prior precipitation step, depth filtration ormembrane filtration can be an effective mechanism for abnormal prionprotein removal from plasma fractions.

[0086] Any acceptable assay that detects prions may be used in thequantitation aspect of the invention. Among these assays are the ELISA,SDS-Page, Western Blot, EG & G Wallac, DELFIA, Prionics assay, Enfer ELCELISA, CEA ELISA, Conformation-dependent assays, DELFIA, and capillaryelectrophoresis, to name a few, all of which are familiar to thosehaving skill in the art.

[0087] The inventors hereof have employed the Western Blot to detectprion from the filtered and eluted biological fluid samples. Westernblotting is a method of used to identify and characterize PrP^(sc). ThePrP^(sc) is isolated by extraction and is differentiated by its partialresistance to proteinase K digestion. The PrP^(RES) (PrP^(sc) resistantto proteinase digestion) is identified by the migration positions of theglycosylation forms and fragments. The sensitivity of this assay isapproximately 3 logs less sensitive than the infectivity assay. Thissensitivity issue is partially overcome by centrifuging the preparation,removing the supernatant and resuspeding the prion material in a smallervolume, resulting in a concentration of the prion material. However,instead of spinning down large volumes of biological fluids such as forexample body fluids, these inventors have shown that the prions can becaptured by treating the biological fluid containing them with anaggregation aid, and then concentrating them by filtering them through afilter and later collecting them in a small volume by elution. Thistechnique can be used on a large scale to remove prions from a productstream.

[0088] This procedure will have a major impact on the use of the Westernblot to determine the presence of PrP^(sc) in a biological matrix. Thisinvention allows the TSE material to be quantitatively concentratedquickly to allow for enhanced detection. When seeking to purify abiological or food solution of PrP^(sc) this invention has the advantagein the ease in which the biological or food solution and materialfilters through the large nominal pore size of the filter.

[0089] The standard Western Blot assay to confirm the specific captureof the prions relies on the captured material first being treated withProteinase K, which digests all normal prion (PrP^(c)) but does notmarkedly digest the abnormal prion (PrP^(sc) or PrP^(res)). The digestis run in accordance with the methods of Lee et al., J Virol Methods2000, 84:77-89, on the SDS gel and transblotted to a sheet ofnitrocellulose or PVDF (polyvinylidene fluoride) membrane. The separatedPrP^(res) bands are then visualized using 3F4 or 6H4. Typical dilutionis 1:2000 for 3F4 (stock 1 mg/ml) or 1:5000 for 6H4 (stock 2.5 mg/ml),10 mL total volume in PBS Tween 20-5% nonfat milk buffer. The antibodiesare detected with goat anti-mouse IgG-HRP conjugate (1:50,000 in thesame buffer). Bands are detected with a HRP substrate usually bechemiluminescence and visualized after exposure to x-ray film. See Leeet al., supra.

[0090] Specific PrP^(sc) monoclonal antibodies like 16A18 canspecifically bind the PrP^(sc) on magnetic beads (Dynal Tosylactivated), Dynal Biotech, Oslo, Norway), and such antibodies can beused to detect presence of PrP^(sc) rather that Western Blot methods.Most of the antibodies in this family can capture PrP^(sc) but detectionhas relied on the 3F4 or 6H4 in a Western Blot format as above.

[0091] Other methods to detect PrP^(sc) include ELISA and SDS-Page andother generally accepted detection methods as disclosed hereinabove.

[0092] In the case where the PrP^(sc) material is captured on a filtersuch as for example a sterilizing filter, which filter specificallybinds prion such as with prion-specific antibody, Western Blot methodsneed not be employed to detect the PrP^(sc). Rather, a prion-specificantibody such as a monoclonal could be employed to detect and quantitatethe prion. Such an antibody includes the generic prion antibodies 6H4 or3F4, which recognize both normal (PrP^(c)) and abnormal (PrP^(sc) andPrP^(res)) prions. If the membrane binds all forms of prion, therelative amount of PrP^(sc) would be very low (for instance less thanabout 1% of prion present). Specific monoclonals for abnormal prions,such as for example 16A18 or 12A5, could be used to detect PrP^(sc) inthe case where the membrane binds all forms (normal and abnormal) ofprions. Using such monoclonals it should be possible to detect PrP^(sc)if the signal could be amplified, if necessary, using chemiluminescencesubstrates or polyHRP conjugates.

[0093] The inventive methods disclosed herein results in an increase inthe detection limit of the assay by greater than 100 fold, nowapproaching the infectivity assay detection limit. Using current methodsavailable in the art, the infectivity assay can take months to yieldresults, dependent upon the species under study. These inventors havealso shown that the assay can be simplified by detecting the presence ofabnormal prion on the membrane surface not requiring elution.

[0094] In the use of the inventive methods of PrP^(sc) aggregation andremoval with an immunoglobulin, and in particular in the manufacture ofan anti-D immunoglobulin, specifically RhoGAM Rho(D) Immune Globulin(Human), and referring to the flowsheet of FIG. 1 and the methods ofCohn et al., J. Am. Chem. Soc., Vol. 68, pages 459-475, thefractionation proceeds from whole human plasma. The plasma is cooled toabout 1° C. and is then centrifuged to separate a cold insolubleprecipitate from a supernatant. The supernatant is further fractionatedto yield Precipitate I and Supernatant I. Precipitate I which consistsprincipally of fibrinogen is discarded. Supernatant I is furtherfractionated to yield Supernatant II+III and Precipitate II+III.Supernatant II+III, which is discarded, contains alpha and beta globulinand lipids. Precipitate II+III consists principally of beta and gammaglobulins and isoagglutinins, but also contains prothrombin,plasminogen, cholesterol and other lipids. Precipitate II+III, uponfurther fractionation yields Supernatant II+III W and PrecipitateII+IIIW. The beta globulin, cholesterol and other lipids are largelyremoved in Supernatant II+III W which is discarded. Precipitate II+III Wconsists principally of gamma globulins, isoagglutinins, plasminogen andprothrombin and some beta globulin, cholesterol and other lipids. Uponfurther fractionation, Precipitate II+III W yields SupernatantIII+Precipitate III. Precipitate III, which is discarded, containsisoagglutinins, plasminogen and prothrombin. Supernatant III consistsprincipally of gamma globulins and minor amounts of fibrinogen andlipids. The final step of the fractionation yields Precipitate II whichis essentially pure gamma G globulin. Precipitate II prepared by theprocess of the invention is an anti-Rh gamma globulin.

[0095] In the preferred methods of the invention, the immunoglobulinstarting material for resuspension is the Precipitate II paste from themodified Cohn process. Lyophilized precipitate II paste may be used ifthe protein is lyophilized in the presence of excipient such as thosecontemplated by U.S. Pat. No. 6,096,872. The filtration process of theinvention to capture prions in this case has preferably already beenperformed in the fractionation of Precipitate II; with reference to theabove and to FIG. 1, the filtration of the Immunoglobulin leading to thecapture of prions is performed before Precipitate II is obtained, afterobtaining Supernatant III, or, most preferably, between Supernatant IIIand Filtered Supernatant III, as shown. Such treatment of material withthe aggregation aid methanol, and at a ratio of about 4 to about 1 MeOH:Supernatant III aggregates prion in the Supernatant III, whichaggregates can then be removed using further methods of the invention.The filtration steps allowing the prion capture of the invention mayalso be done on finished immunoglobulin product. However, treatment withaggregation aid and filtration could also be performed as the finalstage of product processing, so long as the treatment and filtration atthat stage do not interfere with the biological activity or otherwisecompromise the final product.

[0096] The mode of administration of the preparations of the inventionmay determine the sites and/or cells in the organism to which thecompound(s) will be delivered. The compounds purified by the methods ofthe invention can be administered alone but will generally beadministered in admixture with a pharmaceutical carrier or diluentselected with regard to the intended route of administration andstandard pharmaceutical practice. The preparations may be injectedparenterally, for example, intra-arterially or intravenously. Thepreparations may also be delivered via oral, subcutaneous, orintramuscular routes. For parenteral administration, they can be used,for example, in the form of a sterile, aqueous solution which maycontain other solutes, for example, enough salts or glucose to make thesolution isotonic.

[0097] For the oral mode of administration, the purified compositions ofthe invention can be used in the form of tablets, capsules, lozenges,powders, syrups, elixirs, aqueous solutions and suspensions and thelike. In the case of tablets, carriers which can be used includelactose, sodium citrate, and salts of phosphoric acid. Variousdisintegrants such as starch, and lubricating agents such as magnesiumstearate are commonly used in tablets. For administration in capsuleform, useful diluents are lactose and high molecular weight polyethyleneglycols. When aqueous solutions are required for oral use, certainsweetening and/or flavoring agents can be added.

[0098] The substantially pure preparations of the present invention maybe administered to a subject such as a mammal, including humans. Foradministration in the treatment of afflictions, the prescribingphysician or veterinarian will ultimately determine the appropriate dosefor a given human or animal subject, and this can be expected to varyaccording to the weight, age, and response of the individual as well asthe nature and severity of the individual's symptoms.

[0099] In the case of the substantially pure anti-D immunoglobulin ofthe invention, the per-dose dosage will range from about 300 ug forRhoGAM® and about 50 ug for MICRhoGAM®, each of which are administeredin accordance with the guidelines and for the purposes discussedhereinabove and in the respective product literature. Each of theproducts mentioned above can also be multi-dosed, for a total deliveryto be determined by the treating physician.

[0100] The prion-free preparations of the invention may includebiologicals, medicaments, foodstuffs and feeds, and the methods of theinvention may be used in the processing of same.

[0101] Throughout this application, various patents and papers arereferenced. The disclosures thereof in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art as known to those skilled therein as ofthe date of the invention described and claimed herein.

[0102] The following examples are provided for the purposes ofillustration only and are not to be viewed as a limitation of the scopeof the invention.

EXAMPLES Example 1 Production of Rho(D) Immune Globulin Precipitate IIusing Aggregation Aid

[0103] This Example describes a process for the fractionation of humanplasma to obtain Precipitate II to be used in the production of Rho(D)immune globulin.

[0104] Plasma units (anti-D) (a total of approximately 943 L) werestored at 2° C. to 8° C. for four days to allow thawing. The units werepooled in a stainless steel water-jacketed tank through which water at5-10° C. circulates. The pooled plasma was stirred for thirty (30)minutes at 1-3° C. The plasma was then centrifuged in a continuous flowcentrifuge feeding at a rate of 1000 mL/minute. The cold insolublesupernatant (centrifuged plasma) was collected in a stainless steeljacketed tank and stirred until a homogeneous mixture was obtained. Thebatch volume at this point was 905 L of supernatant (clarified plasma).

[0105] The pH of the entire batch of supernatant was adjusted to pH 9.45using 2.172 L of 5.0N NaOH. Methanol (71%), 160.185 L was added to thepH adjusted batch, which was at −5.3° C. pH was 9.37. The batch wasallowed to stand for 13.5 hours at −5.2° C. Final volume was 1067.357L.

[0106] The batch was centrifuged in a continuous flow centrifuge feedingat a rate of 1000 ml/minute at −5.8° C. Supernatant I was collected in astainless steel jacketed tank, and well mixed. Precipitate I wasdiscarded as medical waste. Supernatant I was pH adjusted by adding3.132 L of conc. sodium acetate buffer, pH 4.0 and 627.444 L of 71%methanol. The batch temperature was −5.5° C. and the pH was 6.75. Thebatch was allowed to stand 14 hours.

[0107] The batch was centrifuged in a continuous flow centrifuge feedingat a rate of 1000 ml/minute at −5.5° C. Precipitate II+III wastransferred into a stainless steel pot; 40.220 KG net weight wascollected; this net weight Precipitate II+III was resuspended in twovolumes (L) (80.440L) of Water for Injection, U.S.P. at +1.1° C. andstirred for 45 minutes until a uniform suspension was obtained. Threevolumes (120.660 L of 0.0187M disodium phosphate was added and stirredat 2.1° C. for 30 minutes.

[0108] In a stainless steel jacketed tank, 19 volumes (764.180L) ofWater for Injection, U.S.P. was cooled to 1.0° C. Using a high capacitytransfer pump, the batch was slowly combined with the 19 volumes ofWater for Injection, U.S.P., and was stirred for 30 minutes.

[0109] A volume of 71% methanol was adjusted to equal 15 times theweight of the Precipitate II+III. This methanol (603.300L) was cooled to−14° C. and using a stainless steel Sparger device and a metering pump,the methanol was added to the batch while gradually lowering thetemperature to −5.5° C. The batch was stirred for 1 hour aftercompletion of the methanol addition. pH was 7.23. The batch was allowedto stand for 10 hours 20 minutes.

[0110] Precipitate III was formed via centrifugation of the batch in acontinuous flow centrifuge feeding at a rate of 500 mL/minute at −5.8°C. The Precipitate II+III w was transferred from the bowls into astainless steel pot; the net weight was 22.760 kg.

[0111] The Precipitate II+III w was resuspended in two volumes (L)(45.520L) of Water for Injection, U.S.P. at +1.3° C. and stirred for 45minutes until a uniform suspension was obtained. Two volumes (45.520 L)of 0.175M sodium acetate was added and stirred at 1.4° C. for 30minutes. The pH of the entire batch was adjusted by addition of 0.489Lof sodium acetate buffer, pH 4.0 in 22.760L Water For Injection U.S.P.(total volume 137.049L) to the batch and stirred for 1 hour at 1.5° C.pH was 5.38. In a stainless steel jacketed tank, 13.5 volumes (307.260L)water for Injection U.S.P. was cooled to +2.5° C. with stirring. Using ahigh capacity transfer pump, the batch was combined with the Water forInjection, total calculated volume was 444.309L. NaCl (6.168L of 1.33M)was added to the 22.760 Kg of Precipitate II+III, and was stirred for 30minutes.

[0112] A volume of 71% methanol was adjusted to equal 8.78 times theweight of the Precipitate II+III w. This methanol (199.833L) was cooledto −10.5° C. and using a stainless steel Sparger device and a meteringpump, the methanol was added to the batch while gradually lowering thetemperature to −6.6° C. The batch was stirred for 1 hour aftercompletion of the methanol addition. pH was 5.38. The batch was allowedto stand for 8 hours 30 minutes at −6.3° C.

[0113] Formation of Precipitate III proceeded as follows:

[0114] The batch was centrifuged in a continuous flow centrifuge feedingat a feeding rate of 500 mL/minute at −6.3° C. The Supernatant III wascollected in a stainless steel tank. The Precipitate III was discardedas blood waste.

[0115] The filtration of the Supernatant III proceeded as follows:

[0116] The CUNO filter 90SP housing including (4) 16 sq. ft. cartridges,was assembled in accordance with manufacturer's instruction. Sodiumacetate-methanol buffer (320L) was cooled to −6.5° C., and was filteredthrough the filter cartridges over 55 minutes. The sodiumacetate-Methanol Buffer wash solution was blown completely out of thefilter cartridge before proceeding. The batch was filtered using theCuno filter 90SP in accordance with good manufacturing practice andemploying manufacturer's instructions. When the entire volume ofSupernatant III was filtered, the pressure in the filter housing wasreleased. Volume of filtered Supernatant III was 622L, and was stirredat moderate speed. NaCl (1.33M, 23.387 mL) was added to Supernatant IIIslowly and stirred for 30 minutes at 6.5° C. pH was 5.38, and adjustedto 7.10 with 4.840L of 1.0 M sodium bicarbonate and mixing for 30minutes. Methanol (100%) equal to 0.166 times the volume of SupernatantIII (103.252 L) was added to the Supernatant III using a Sparger deviceand a metering pump and the batch stirred vigorously. PH was 7.3.

[0117] Fractionation of Precipitate II was performed as follows: Thebatch was centrifuged in a continuous flow centrifuge feeding at a rateof 500 mL/minute at −6.3° C. and the supernatant discarded. Dry nitrogenwas used to blow out the feed lines and dry spun for 15 minutes. ThePrecipitate II (7,420 g) was transferred from the centrifuge bowls intoa tared, stainless steel pot and stored at −22.1° C. This material wasused in the viral clearance process in accordance with the methods ofco-assigned U.S. Patent to Van Holten et al., U.S. Pat. No. 6,096,872issued Aug. 1, 2000.

Example 2 Elution and Detection of Prions from Example 1

[0118] The prions collected on the Cuno depth filter used to filter theSupernatant III obtained by the methods of Example 1 hereinabove areeluted, quantitated and detected using the methods of Example 3hereinbelow.

[0119] In particular, the Cuno depth filter pad used in Example 1 isremoved from the filter housing and placed in a petri dish with 45 ml of1.0M NaCl (elution buffer). The petri dish is placed on a rotary shakerand swirled gently for about 20-30 minutes. The filter is removed andsimilarly washed a second time with 45 mL 10 of 2.0M NaCl (elutionbuffer) for 20-30 minutes.

[0120] Western Blot analysis of PrP^(sc) on the eluate is performed oneluate from the 1.0M NaCl elution buffer and a second Western Blotperformed separately on the eluate from the 2.0M NaCl elution, both inaccordance with the Western Blot methods of Example 3.

Example 3 Removal and Quantitation of PrP^(sc) from ImmunoglobulinPreparation

[0121] Supernatant III (SupIII) (190 mL) containing anti-D was obtainedfrom a full-scale (approx. 450 Liters) modified Cohn-Oncleyfractionation (Ortho-Clinical Diagnostics, Raritan N.J.) (See Example 1hereinabove). The SupIII was stored at −70° C. and thawed at 25° C. justprior to the addition of the scrapie brain homogenate (SBH), thenequilibrated at a temperature of at −5.5 to −7.5° C.

[0122] Brain Homogenate

[0123] Scrapie brain homogenate (10%) was prepared using brain fromhamsters infected with 263K hamster-adapted agent. Frozen brains(approx. 3-20 as ˜0.5 grams per brain) were thawed on ice, thenhomogenized in nine volumes of Tris buffered saline, pH 8.0. Thehomogenate was clarified by centrifugation at 1200 r.c.f. at 2-80 C for20 minutes. One percent (1%) lysolecithin was added to the supernatantto a final concentration of 0.1% (w/v). This material was stored at −70°C. until use. Prior to use, the SBH was thawed in a room temperaturewater bath, then cup horn-sonicated (Misonix Sonicator XL2020 with cuphorn, (Heat Systems, Farmingdale, N.Y.) for approximately two minutesper milliliter until the solution turned from turbid to translucent. Thetreated homogenate was then serially filtered through Millex® 25 mm PVDFsyringe-driven filter units, (Millipore Corporation) 0.45/0.22/0.1micron filters, which further clarified the material. The SupernatantIII (SupIII) (200 mL) from the Cohn fractionation process was spikedwith the filtered SBH (1:51 dilution). The second run was filteredthrough a 0.22 micron filter just prior to the start of the depthfiltration to remove any aggregates that may have formed in the mixture(see Example 5). Samples of SHB were sampled for Western blot evaluationprior to treatment and after sonication and filtration.

[0124] Filtration

[0125] A 47 mm CUNO Zeta Plus 90SP filter pad (Cuno Corporation, MeridanConn.) was placed in its stainless steel filter housing. A peristalticpump was used to control the flow rate of the filtration to a rate ofabout 1 ml/min. The entire filter housing was placed in an insulatedsodium chloride ice bath to cool the filter to approx. −5.5 to −7.5° C.Sodium acetate-methanol buffer (80 ml of 0.01N sodium acetate methanolbuffer, 22.7% MeOH at −5.5 to −7.5° C. was used to wash the filter. TheSBH-spiked SupIII (180 ml) at −5.5 to −7.5° C was filtered through theCUNO filter at a flow rate of 1.0 mL/minute. Aliquots of filtrate werecollected at the beginning (75 ml), middle (75 ml) and end (30 ml) ofthe filtration. The pressure of the system was monitored during theentire filtration and was about 2 psi.

[0126] Elution of PrP_(sc) from Filter

[0127] After filtration, the filter pad was removed from the filterhousing and placed, rough side up, into a beaker and washed with 45 mLof 1.0M NaCl elution buffer for 20-30 minutes by gently swirling on arotary shaker. The filter was removed and washed a second time with 45mL of 2.0M NaCl for 20-30 minutes with gentle swirling on the rotaryshaker. It would be possible to further concentrate the PrP^(sc) bycentrifugation at 100,000×g for about 1 hr. at 4 degrees C., howeverthis was not necessary as it was sufficiently concentrated for WesternBlot analysis as shown in Table 1.

[0128] A second run was identical to the first, except that the SupIIIspiked with SBH was first pre-filtered through a Millex 0.22 micronfilter.

[0129] A control run was performed, cooling the filter apparatus to 0°C. and washing the depth filter pad with 80 mL of TBS. TBS (180 mL)spiked with filtered SBH (1:51 dilution) was filtered under the sameflow rates as above, followed by the filter washes. See Example 4.

[0130] Western Blot analysis of PrP^(sc) on the eluate was performed oneluate from the 1.0M NaCl elution buffer and a second Western Blotperformed on the eluate from the 2.0M NaCl elution, both in accordancewith the Western Blot methods hereinbelow.

[0131] With reference to Table 1, data is shown wherein PrP^(sc) ispresent as having been eluted from the filter after both elutions.

[0132] Western Blot

[0133] Sample Preparation

[0134] Sample preparation and assay methodology was performed inaccordance with Lee et al., J Virol Methods 2000; 84:77-89. Samples weretreated in a proteinase K digestion step that is used to differentiatethe PrP^(c) from the PrP^(sc). Following the proteinase K treatment thesamples were centrifuged at 20,000 r.c.f. for 1 hour at 4° C. Thepellets were resuspended in 10 μl each of 2× sodium dodecyl sulfate(SDS) sample buffer and heated at 100° C. for five minutes. Half-logserial dilutions were prepared prior to loading onto gels for thedetection of the PrP^(RES) by Western blot, all in accordance with Leeet al. (supra)

[0135] Assay

[0136] Samples were assayed according to the method of Lee et al.,supra. Each sample was electrophoresed on a 12% SDS-Tris-glycinepolyacrylamide gel for 60 minutes at 125 constant volts. Gels weretransferred to nitrocellulose membranes for 60 minutes at 125 constantmA, then soaked in TBS and blocked for 60 minutes in 5% non-fat milk.Following transfer and blocking the membrane was incubated in 3F4monoclonal antibody. After washing, the membrane was exposed to analkaline phosphatase-conjugated anti-mouse IgG secondary antibody. Theblot was then soaked in CDP-Star plus NitroBlock II, and then exposed toKodak XAR-2 film. A valid test was determined by the positive controlexhibiting banding at 33 kDa mark. Two smaller less intense bands thanthe 33 kDa band are also typically observed. This triplet of bands istypical Western blot image for PrP^(RES) (Lee et al, supra.).

[0137] Results

[0138] Sonication and serial membrane filtration removed all turbidityfrom the SBH. The subsequent depth filtration of the SBH spikedimmunoglobulin preparation reduced the PrP^(sc) concentration in thefiltrate to a level below the limits of detection of the Western blotassay (Table 1) A significant amount of the PrP^(sc) was recovered fromthe filter pad by elution with high salt solutions. Filtration of theSBH spiked SupIII through a 0.22 micron filter prior to depth filtrationremoved PrP^(sc) to undetectable levels. The depth filtration of the SBHspiked into the buffer control removed little or no PrP^(sc).

[0139] In Example 3 herein, membrane filtration of the sonicated SBH wasperformed prior to depth filtration of the SBH spiked SupIII in order toinsure that the depth filter would see particles no greater than 0.1micron in size. This would present the greatest challenge to the depthfilter and would allow characterization of the mechanism of PrP^(sc)removal. The SBH was first sonicated to break up the PrP^(sc) aggregatesand facilitate the membrane filtration. Despite the sonication, it wasnecessary to serially filter the SBH through progressively smallerfilters (0.45 and 0.22 micron) to minimize clogging of the 0.1 micronfilter.

[0140] Examination of the depth filter after filtration of the SBHspiked SupIII and prior to elution with the 1.0M and 2.0M NaCl solutionsrevealed a small amount of material on the surface of the depth filter.This was believed to be a precipitate formed when the SBH was added tothe SupIII, caused by the methanol present in the SupIII. In order todetermine if this precipitate contained PrP^(sc), a second run wasperformed where the SBH spiked SupIII was first pre-filtered through a0.22 micron filter prior to depth filtration. See Example 6. Thepre-filtration removed PrP^(sc) to undetectable levels, indicating thatin the prior run the PrP^(sc) was removed by precipitation andmechanical straining, rather than by adsorption to the depth filter.TABLE 1 Determination of PrP^(SC) by Western blot assay in an ImmuneGlobulin preparation spiked with scrapie brain homogenate (SBH) TotalLog₁₀ (prion unit) Mass Log₁₀ Sample Name Reduction Balance Factor*Depth Filtration of SBH spiked SupIII Spiked Load 6.9 100%  >5.2 EarlyFiltrate <2.7 0% Middle Filtrate <2.1 0% Late Filtrate <2.1 0% SaltStrip (1 M) 5.2 32%  Salt Strip (2 M) 5.0 1% Depth Filtration of SBHspiked SupIII with prior 0.22 μm filtration Spiked Load 7.1 100%  >3.0Spiked Load II <4.1 0% (0.22 μm filtered)† 0% Early Filtrate <2.7 0%Middle Filtrate <2.1 0% Late Filtrate <2.0 0% Salt Strip (1 M) 3.2 0%Salt Strip (2 M) <3.5 0% Depth Filtration of SBH spiked TBS Spiked Load7.0 100% 0.8 Early Filtrate 6.2  16% Middle Filtrate 6.7  50% LateFiltrate 6.0  11% Salt Strip (1 M) 4.5  0.3% Salt Strip (2 M) 4.5  0.3%

Example 4 Control

[0141] In order to determine whether depth filtration would removePrp^(sc) in the absence of a precipitating alcohol, a control run wasperformed where the PrP^(sc) was spiked into an aqueous buffer and thendepth filtered.

[0142] The materials and procedures of Example 3 were repeated whereinthe same concentration and volume (3.6 ml) of SBH was spiked into 180 mlof 0.1 M Tris Buffered Saline (TBS). The lack of removal of PrP^(sc)from the buffer control indicated that the depth filter did not retainthe protein, either by mechanical means (because the PrP^(sc) hadpreviously passed through a 0.1 micron filter) nor by electrokineticadsorption. See Table 1.

[0143] These data indicate that previous reports on the effectiveness ofdepth filtration to remove PrP^(sc) may be misleading. Indeed, depthfiltration does remove PrP^(sc), not by the absorptive mechanism usuallyassociated with depth filtration but by mechanical straining of theprecipitated protein. The results of this study indicate that depthfiltration alone is ineffective in removing PrP^(sc). However, when usedin conjunction with a prior precipitation step, depth filtration ormembrane filtration can be an effective mechanism for abnormal prionprotein removal from plasma fractions.

Example 5 Elution of PrP^(sc) from Filter

[0144] The filter pad used in Example 3 was removed from the filterhousing and placed in a petri dish with 45 ml of 1.0M NaCl (elutionbuffer). The petri dish was placed on a rotary shaker and swirled gentlyfor about 20-30 minutes. The filter was removed and similarly washed asecond time with 45 mL of 2.0M NaCl (elution buffer) for 20-30 minutes.

[0145] Western Blot analysis of PrP^(sc) on the eluate was performed oneluate from the 1.0M NaCl elution buffer and a second Western Blotperformed separately on the eluate from the 2.0M NaCl elution, both inaccordance with the Western Blot methods of Example 3. With reference toTable 1, data is shown wherein PrP^(sc) is present as having been elutedfrom the filter after both elutions.

Example 6 Pre Filtration of SBH in 0.22 Micron Filter

[0146] In order to determine if the precipitate observed on the filterprior to the depth filtration step of Example 3 contained PrP^(sc), asecond run was performed where the SBH spiked SupIII was firstpre-filtered through a 0.22 micron filter prior to depth filtration. Thematerials and procedures of Example 3 were repeated wherein the SBHspiked SupIII was pre-filtered through a 0.22 micron filter prior todepth filtration. With reference to Table 1, it was demonstrated thatthe pre-filtration removed PrP^(sc) to undetectable levels, indicatingthat in the prior run the PrP^(sc) was removed by precipitation andmechanical straining, rather than by electrostatic interaction with thedepth filter.

Example 7 Elution of PrP^(sc) from Filter

[0147] Filter pads used in Example 6 were removed from the filterhousing and placed in a petri dish with 45 ml of 1.0M NaCl elutionbuffer. The petri dish was placed on a rotary shaker and swirled gentlyfor about 20-30 minutes. The filter was removed and similarly washed asecond time with 45 mL of 2.0M NaCl elution buffer for 20-30 minutes.

[0148] Western Blot analysis of PrP^(sc) on the eluate was performed oneluate from the 1.0M NaCl elution buffer and a second Western Blotperformed separately on the eluate from the 2.0M NaCl elution, both inaccordance with the Western Blot methods of Example 3.

[0149] With reference to Table 1, data is shown wherein PrP^(sc) ispresent as having been eluted from the filter after both elutions.

Example 8 Clearance of Prions from Blood Sample

[0150] Cow whole blood (250 ml) is centrifuged at 100×g to remove thered cells. The resulting plasma is admixed with 75 ml of 22.7% methanolto aggregate the prion material. The admixture is gently swirled for 5minutes on a rotary mixer. The admixture is passed through a 47 mm CunoZeta Plus 90S filter that was prepared as in Example 3 hereinabove. Thematerial is then eluted for Western Blot assay by washing the filter padin 5 ml of 1.0 M NaCl-15 mg/mL glycine solution. Following extractionand concentration in accordance with Lee et al., 0.5 ml of this materialwas analyzed by Western Blot in accordance with the methods of Example3.

[0151] The above procedure results in an increase in the detection limitof the assay by greater than 100 fold, now approaching the infectivityassay detection limit. Using current methods available in the art, theinfectivity assay can take months to yield results, dependent upon thespecies under study. These inventors have also shown that the assay canbe simplified by detecting the presence of abnormal prion on themembrane surface not requiring G17 elution.

Example 9 Clearance of Prions from Urine Sample

[0152] A human urine sample (200 ml) is sedimented for 5 minutes at 3000rpm to discard occasional cell debris. The urine sample is admixed with75 ml of 22.7% methanol to aggregate the prion material. The admixtureis gently swirled for 5 minutes on a rotary mixer. The admixture ispassed through a 47 mm Cuno Zeta Plus 90S filter that was prepared as inExample 3 hereinabove. The material is then eluted for Western Blotassay by washing the filter pad in 5 ml of 1.0 M NaCl-15 mg/mL glycinesolution. Following extraction and concentration in accordance with Leeet al., 0.5 ml of this material is analyzed by Western Blot inaccordance with the methods of Example 3.

[0153] It will be understood by those skilled in the art that theforegoing description and examples are illustrative of practicing thepresent invention, but are in no way limiting. Variations of the detailpresented herein may be made without departing from the scope and spiritof the present invention.

We claim:
 1. A method of removing prion protein from an aqueous liquidcontaining biological or food product, comprising: (a) admixing theaqueous liquid with one or more aggregation aids; and (b) filtering theadmixture of step (a) through a filter thereby removing the prionprotein.
 2. The method of claim 1 wherein the prion protein is normalprion protein, abnormal infective prion protein, or a mixture of normaland abnormal infective prion protein.
 3. The method of claim 2 whereinthe biological product is selected from the group containing wholeblood, blood components, urine, CSF, liquids containing albumin,immunoglobulins and fragments thereof, blood coagulation factors such asFactor IX, thrombin, fibronectin, fibrinogen, Factor VIII, II, VII, IX,X, XI, XIII, hemoglobin, alpha-2-macroglobulin, haptogobin, transferrin,apolipoproteins, protein C, protein S, C-1-esterase inhibitor, enzymes,inter-alpha-trypsin inhibitor, growth hormones and Von Willebrandfactor.
 4. The method of claim 1 wherein the food comprises foods anddrinks.
 5. The method of claim 3 wherein the blood components compriseserum and plasma.
 6. The method of claim 3 wherein the immunoglobulin ispolyclonal or monoclonal.
 7. The method of claim 6 wherein theimmunoglobulin is IgG.
 8. The method of claim 7 wherein the IgGimmunoglobulin is IgG anti-D immunoglobulin.
 9. The method of claim 8wherein the IgG immunoglobulin of claim 8 is in a pharmaceuticalcomposition comprising from about 4.0 to 6.0% immunoglobulin by weight,and from about 80 to 200 ppm polysorbate
 80. 10. The method of claim 3wherein the one or more aggregation aids are admixed together or used inseries.
 11. The method of claim 10 wherein the one or more aggregationaids comprise organic solvents of low dielectric constant.
 12. Themethod of claim 11 wherein the organic solvents are selected from thegroup consisting of acetone and water-miscible alcohols.
 13. The methodof claim 11 wherein the organic solvents are selected from the groupconsisting of ethanol, methanol, isopropyl, isopropanol, n-propanol,isopropyl ether, ketones and aldehydes.
 14. The method of claim 13wherein the alcohol is ethanol or methanol at a concentration of fromabout 2% to about 100%.
 15. The method of claim 10 wherein the one ormore aggregation aids are selected from the group containing ammoniumsulfate, caprylic acid, trichloroacetic acid (TCA), dialdehydes,heteropoly acids, lactate monohydrate (C18H21N3O4H2O), and the metalions Cu2+, Ni, Zn and Ag.
 16. The method of claim 10 wherein when theprion protein comprises abnormal prion protein, the one or moreaggregation aids are complexing agents.
 17. The method of claim 16wherein the complexing agent is selected from the group containingheteropolymolybdates, heteropolytungstates, sodium phosphotungstate(NaPTA), antibodies, enzymes and peptides.
 18. The method of claim 10wherein the filter is a membrane or depth filter.
 19. The method ofclaim 18 wherein the filter is a depth filter.
 20. The method of claim18 wherein the filter has a pore size providing a retention of less thanabout 6 μm.
 21. The method of claim 20 wherein the filter has a poresize providing a retention of about 0.6 to about 1.5 microns.
 22. Themethod of claim 19 wherein the depth filter has a pore size providing aretention of less than about 0.6 microns.
 23. The method of claim 22wherein the recovery of the biological protein in its originalbiological state is substantially maintained at least to a level inexcess of about 50%.
 24. The method of claim 22 wherein the abnormalinfective prion protein may be achieved to an extent of at least about10^(2.5).
 25. A substantially pure pharmaceutical composition comprisingan abnormal infective prion-cleared immunoglobulin for injection inaccordance with the method of claim
 24. 26. The substantially pureimmunoglobulin of claim 25 wherein the immunoglobulin is IgG anti-Dimmunoglobulin.
 27. The substantially pure IgG anti-D immunoglobulin ofclaim 26, wherein the IgG anti-D immunoglobulin is in a pharmaceuticalcomposition comprising from about 4.0 to 6.0% immunoglobulin by weight,and from about 80 to 200 ppm polysorbate
 80. 28. A method of removingprion protein from whole blood, comprising: (a) clinically centrifugingthe blood to separate the red blood cells and platelets therefrom; (b)decanting supernatant of the centrifugation of step (a) from the redblood cells and platelets; (c) admixing the supernatant with one or moreaggregation aids; (d) filtering the admixture of step (c) through amembrane or depth filter, thereby removing the prion protein fromfiltrate; and (e) adding the red blood cells and platelets separated instep (a) back to the filtrate.
 29. The method of claim 28 wherein theaggregation aids are admixed together or used in series.
 30. The methodof claim 29 wherein the one or more aggregation aids comprise organicsolvents of low dielectric constant.
 31. The method of claim 30 whereinthe organic solvents are selected from the group consisting of acetoneand water-miscible alcohols.
 32. The method of claim 30 wherein theorganic solvents are selected from the group consisting of ethanol,methanol, isopropyl, isopropanol, n-propanol, isopropyl ether, ketonesand aldehydes.
 33. The method of claim 32 herein the alcohol is ethanolor methanol at a concentration of from about 2% to about 100%.
 34. Themethod of claim 29 wherein the one or more aggregation aids are selectedfrom the group containing ammonium sulfate, caprylic acid,trichloroacetic acid (TCA), dialdehydes, heteropoly acids, lactatemonohydrate (C18H21N3O4H2O), and the metal ions Cu2+, Ni, Zn and Ag. 35.The method of claim 28 wherein when the prion protein comprises abnormalprion protein, the one or more aggregation aids are complexing agents.36. The method of claim 35 wherein the complexing agent is selected fromthe group containing heteropolymolybdates, heteropolytungstates, sodiumphosphotungstate (NaPTA), antibodies, enzymes and peptides.
 37. Themethod of claim 28 wherein the filter is a membrane or depth filter. 38.The method of claim 37 wherein the filter is a depth filter.
 39. Themethod of claim 38 wherein the filter has a pore size providing aretention of less than about 6 μm.
 40. The method of claim 39 whereinthe filter has a pore size providing a retention of about 0.6 to about1.5 microns.
 41. The method of claim 38 wherein the depth filtration iscarried out using a using a depth filter having a pore size providing aretention of less than about 0.6 microns.
 42. A method for the capture,elution, concentration, and quantitation of abnormal infective prionprotein associated with TSEs in an aqueous solution of a biological orfood product, comprising: (a) admixing the aqueous liquid with one ormore aggregation aids; (b) filtering the admixture of step (a) through afilter thereby removing the prion protein; (c) eluting the prions fromthe filter; and (d) quantitating the abnormal infective prion proteinusing an assay.
 43. The method of claim 42 wherein the biologicalproduct is selected from the group containing whole blood, bloodcomponents, urine, CSF, liquids containing albumin, immunoglobulins andfragments thereof, blood coagulation factors such as Factor IX,thrombin, fibronectin, fibrinogen, Factor VIII, II, VII, IX, X, XI,XIII, hemoglobin, alpha-2-macroglobulin, haptogobin, transferrin,apolipoproteins, protein C, protein S, C-1-esterase inhibitor, enzymes,inter-alpha-trypsin inhibitor, growth hormones and Von Willebrandfactor.
 44. The method of claim 42 wherein the food comprises foods anddrinks.
 45. The method of claim 43 wherein the blood components compriseserum and plasma.
 46. The method of claim 43 wherein the immunoglobulinis polyclonal or monoclonal.
 47. The method of claim 46 wherein theimmunoglobulin is IgG.
 48. The method of claim 47 wherein the IgGimmunogloblulin is IgG anti-D immunoglobulin.
 49. The method of claim 48wherein the IgG anti_d immunoglobulin of claim 48 is in a pharmaceuticalcomposition comprising from about 4.0 to 6.0% immunoglobulin by weight,and from about 80 to 200 ppm polysorbate
 80. 50. The method of claim 42wherein the one or more aggregation aids are admixed together or used inseries.
 51. The method of claim 50 wherein the one or more aggregationaids comprise organic solvents of low dielectric constant.
 52. Themethod of claim 51 wherein the organic solvents are selected from thegroup consisting of acetone and water-miscible alcohols.
 53. The methodof claim 51 wherein the organic solvents are selected from the groupconsisting of ethanol, methanol, isopropyl, isopropanol, n-propanol,isopropyl ether, ketones and aldehydes.
 54. The method of claim 53herein the alcohol is ethanol or methanol at a concentration of fromabout 2% to about 100%.
 55. The method of claim 50 wherein the one ormore aggregation aids are selected from the group containing ammoniumsulfate, caprylic acid, trichloroacetic acid (TCA), dialdehydes,heteropoly acids, lactate monohydrate (C18H21N3O4H2O), and the metalions Cu2+, Ni, Zn and Ag.
 56. The method of claim 50 wherein when theprion protein comprises abnormal prion protein, the one or moreaggregation aids are completing agents.
 57. The method of claim 56wherein the complexing agent is selected from the group containingheteropolymolybdates, heteropolytungstates, sodium phosphotungstate(NaPTA), antibodies, enzymes and peptides.
 58. The method of claim 50wherein the filter is a membrane or depth filter.
 59. The method ofclaim 58 wherein the filter is a depth filter.
 60. The method of claim58 wherein the filter has a pore size providing a retention of less thanabout 6 μm.
 61. The method of claim 60 wherein the filter has a poresize providing a retention of about 0.6 to about 1.5 microns.
 62. Themethod of claim 59 wherein the depth filter has a pore size providing aretention of less than about 0.6 microns.
 63. The method of claim 62wherein the recovery of the biological protein in its originalbiological state is substantially maintained at least to a level inexcess of about 50%.
 64. The method of claim 62 wherein the abnormalinfective prion protein may be achieved to an extent of at least10^(2.5).
 65. The method of claim 42 wherein the eluting comprisesgentle washing of the filter with an elution buffer.
 66. The method ofclaim 65 wherein the elution buffer comprises a hypertonic solution. 67.The method of claim 66 wherein the hypertonic solution comprises ahypertonic salt solution.
 68. The method of claim 67 wherein thehypertonic salt solution is selected from the group consisting of1.0-2.0M NaCl buffers, and sodium acetate-methanol buffers atconcentrations of about 1.0M to about 2.0M.
 69. The method of claim 42additionally comprising an additional step between step (c) and step(d), comprising concentrating the prions in the elution buffer in anamount suitable for quantitating using currently available assays. 70.The method of claim 69 wherein the concentrating step between step (c)and step (d) comprises centrifuging the prions in the elution buffer.71. The method of claim 42 wherein the quantitating step (d) comprisesusing an available assay selected from the group consisting of ELISA,SDS-Page, Western Blot, EG & G Wallac, DELFIA, Prionics assay, Enfer ELCELISA, CEA ELISA, Conformation-dependent assays, DELFIA, and capillaryelectrophoresis.
 72. The method of claim 42 wherein the quantitatingstep (d) comprises using an antibody assay using prion antibodies 6H4,3F4, 16A18 or 12A5.
 73. A method of removing prion protein from aaqueous pharmaceutical composition comprising an IgG anti-Dimmunoglobulin, wherein the pharmaceutical composition comprises fromabout 4.0 to 6.0% immunoglobulin by weight, and from about 80 to 200 ppmpolysorbate 80, comprising: (a) admixing the aqueous composition withfrom about 2% to about 10% methanol; and (b) filtering the admixture ofstep (a) through a depth filter having a pore size providing a retentionof less than about 0.6 microns, thereby removing the prion protein,wherein the recovery of the biological protein in its originalbiological state is substantially maintained at least to a level inexcess of about 50%, and wherein the abnormal infective prion proteinmay be achieved to an extent of at least about 10^(2.5.)
 74. Asubstantially pure pharmaceutical composition comprising an abnormalinfective prion-cleared immunoglobulin for injection in accordance withthe method of claim
 73. 75. A method of removing prion protein fromwhole blood, comprising: (a) clinically centrifuging the blood toseparate the red blood cells and platelets therefrom; (b) decantingsupernatant of the centrifugation of step (a) from the red blood cellsand platelets; (c) admixing the supernatant with methanol; (d) filteringthe admixture of step (c) through a depth filter having a pore sizeproviding a retention of about 0.6 to about 1.5 microns, therebyremoving the prion protein from filtrate; and (e) adding the red bloodcells and platelets separated in step (a) back to the filtrate.